Wright Penny A, Boyd Helen F, Bethell Richard C, Busch Michael, Gribbon Phillip, Kraemer Joachim, Lopez-Calle Eloisa, Mander Thomas H, Winkler Dirk, Benson Neil
Pfizer Global Research and Development, Sandwich, Kent, United Kingdom.
J Biomol Screen. 2002 Oct;7(5):419-28. doi: 10.1177/108705702237673.
This paper describes the development of a robust, miniaturizable, and quantitative fluorescence-based assay for mitogen-activated protein kinase kinase 7 (MKK7). As a first step, the basic steady-state kinetics of the MKK7-catalyzed phosphorylation of c-Jun N-terminal kinases (JNKs) 1, 2, and 3 were defined using standard radiometric methods. Subsequently, the authors found that in addition to the holo JNKs, a series of novel small peptides (based on the region around the JNK phosphorylation site) are also substrates, provided that these were prephosphorylated on the Y residue of the TPY motif. One of these peptide substrates was used in the development of a fluorescence polarization-based assay using an antibody as a sensor. The assay was successfully miniaturized for use with conventional fluorescence polarization (FP) reader technology in 8.5 microl and on the single microl scale using Evotec proprietary 2-dimensional fluorescence intensity distribution analysis (2D-FIDA) anisotropy and liquid handling technology. The steady-state kinetic parameters derived using the FP or 2D-FIDA anisotropy format assays correlated well with those generated using a radiometric assay. Moreover, the quantitative sensitivity to known inhibitors was maintained independent of the format and assay volume. In addition, the authors found that the 2D-FIDA anisotropy assay exhibited superior performance statistics (typical Z' = approximately 0.5) relative to conventional FP (typical Z' = 0.3) and yielded the additional benefit of order-of-magnitude savings in terms of reagent costs. The 2D-FIDA anisotropy assay was used to carry out a successful high-throughput screening in 1-microl final volume against company file compounds.
本文描述了一种用于丝裂原活化蛋白激酶激酶7（MKK7）的稳健、可小型化且基于荧光的定量检测方法的开发。第一步，使用标准放射性方法确定了MKK7催化c-Jun氨基末端激酶（JNK）1、2和3磷酸化的基本稳态动力学。随后，作者发现，除了全长JNK外，一系列新型小肽（基于JNK磷酸化位点周围区域）也是底物，前提是这些小肽在TPY基序的Y残基上预先磷酸化。其中一种肽底物被用于开发一种基于荧光偏振的检测方法，使用抗体作为传感器。该检测方法成功实现了小型化，可与传统荧光偏振（FP）读数器技术配合使用，检测体积为8.5微升，并且使用Evotec专有的二维荧光强度分布分析（2D-FIDA）各向异性和液体处理技术可在单微升规模上进行检测。使用FP或2D-FIDA各向异性格式检测得出的稳态动力学参数与使用放射性检测得出的参数相关性良好。此外，对已知抑制剂的定量敏感性不受检测格式和检测体积的影响。此外，作者发现，相对于传统FP（典型Z' = 0.3），2D-FIDA各向异性检测表现出更优的性能统计数据（典型Z' = 约0.5），并且在试剂成本方面节省了一个数量级。2D-FIDA各向异性检测用于在1微升最终体积下对公司档案化合物进行成功的高通量筛选。
